Decoy peptides inhibiting protein phosphatase 1-mediated dephosphorylation of phospholamban

ABSTRACT

The present invention relates to decoy peptide or polypeptide consisting of a peptide sequence represented by the following Formula I: X1-Ala-X2—X3-Ile-Glu-X4 (I). It is noteworthy that the decoy peptide or polypeptide of the present invention significantly elevates phosphorylation levels of PLB by inhibiting PP1-mediated dephosphorylation. In addition, the decoy peptide or polypeptide provides cardio-protective effects by restoring of SERCA2a activity and inotropic effect of enhancing myocardial contractility. The present invention will contribute greatly to the prevention or treatment of diseases associated with PLB.

CROSS REFERENCE TO RELATED APPLICATIONS AND CLAIM OF PRIORITY

This application is a National Stage entry from InternationalApplication No. PCT/KR2013/006009, filed Jul. 3, 2013, which claimspriority from U.S. Provisional Patent Application No. 61/668,034 filedon Jul. 5, 2012, entire contents of which are incorporated herein byreference.

STATEMENT AS TO FEDERALLY FUNDED RESEARCH

This invention was supported by the Global Research Laboratory Program(M6-0605-00-0001) funded by the Korean Government (MEST), a grant fromthe Systems Biology Infrastructure Establishment Grant provided by GIST,and NIH grant (HL-080498-01).

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to a decoy peptide or polypeptide ofphospholamban capable of inhibiting the protein phosphatase 1-mediateddephosphorylation of phospholamban and its use as well as a method forpreparing the decoy peptide or polypeptide.

Description of the Related Art

Heart failure remains a leading cause of mortality and morbidityworldwide [1-3]. It is characterized by an increased ventricular chambersize and reduced systolic function of the heart. Previous studiessupport the notion that abnormalities in cardiac contractility cause theinitiation and progression of heart failure [4-6]. The contractility ofcardiomyocytes is directly regulated by intracellular Ca²⁺ cycling [7,8]. A small amount of extracellular Ca²⁺ enters cardiomyocytes throughthe voltage-dependent L-type Ca²⁺ channel and is then followed by alarge release of Ca²⁺ from the sarcoplasmic reticulum (SR) through theryanodine receptor (RyR) [9]. This increase in intracellular Ca²⁺triggers contraction of the myofilaments. Re-uptake of Ca²⁺ back intothe SR through the SR Ca²⁺-ATPase (SERCA) 2a and the Na⁺/Ca²⁺ exchangerat the sarcolemma then initiates relaxation of the myofilaments.

Previous studies showed that decreased SERCA2a expression and activityare associated with heart failure in humans and animal models [10-12].Therefore, the restoration of SERCA2a levels by increasing the genedosage was thought to be a rational approach for the treatment of heartfailure. This proved to be the case when using heart failure models ofrats [13-15] and pigs [16]. Furthermore, adeno-associated virus-mediateddelivery of SERCA2a was recently shown to be a safe and effectivemodality for improving the cardiac functions in heart failure patients[17, 18].

The activity of SERCA2a is negatively regulated by an endogenousinhibitor, PLB (phospholamban), which in turn is regulated by PKA(protein kinase A), CaMKII (Ca²⁺/calmodulin-dependent protein kinaseII), and PP1 (protein phosphatase 1). Phosphorylation at Ser¹⁶ and Thr¹⁷of PLB by PKA and CaMKII, respectively, causes the disassociation of PLBfrom SERCA2a, permitting near-maximal Ca²⁺-ATPase activity of SERC2a[19-21]. On the contrary, dephosphorylation of PLB at Ser¹⁶ or Thr¹⁷ byPP1 enhances the association between PLB and SERCA2a and the inhibitionof SERCA2a by PLB [22, 23]. Intriguingly, reduced PLB phosphorylation[24-26] concomitantly with an increased PP1 activity [27, 28] has beenobserved in animal models and end-stage human heart failure. Therefore,normalization of PP1 activity and PLB phosphorylation would be areasonable approach to enhance cardiac function and suppress theprogression of heart failure.

Molecular decoys such as small peptides that mimic target proteins havebeen successfully utilized to interfere with protein-proteininteractions [29], phosphorylation of target proteins by protein kinases[30], and dephosphorylation of phosphorylated proteins by phosphatases[31, 32].

Throughout this application, various patents and publications arereferenced, and citations are provided in parentheses. The disclosure ofthese patents and publications in their entities are hereby incorporatedby references into this application in order to more fully describe thisinvention and the state of the art to which this invention pertains.

SUMMARY OF THE INVENTION

The present inventors have made intensive researches to a decoy peptideor polypeptide for treating diseases associated with PBL, in particular,caused by decreased SERCA2a activity by dephosphorylation levels of PLB.As a result, we have synthesized a decoy peptide which significantlyelevates phosphorylation levels of PLB. In addition, we have discoveredthat the decoy peptide increases contractile parameters in vitro andimproves left ventricular developed pressure ex vivo. That is to say, wehave found out that the decoy peptide or polypeptide providescardio-protective effects by restoring of SERCA2a activity and inotropiceffect of enhancing myocardial contractility and thus the decoy peptideor polypeptide would be used to treat the diseases associated with PLBby inhibiting dephosphorylation of PLB.

Accordingly, it is an object of this invention to provide a decoypeptide or polypeptide inhibiting PP1-mediated dephosphorylation of PLBby a competitive inhibition.

It is another object of this invention to provide a pharmaceuticalcomposition for preventing or treating diseases associated with PLB.

It is still another object of this invention to provide a method forpreparing the decoy peptide or polypeptide of PLB capable of inhibitingPP1-mediated dephosphorylation of PLB by a competitive inhibition.

It is further object of this invention to provide a method forpreventing or treating diseases associated with PLB.

Other objects and advantages of the present invention will becomeapparent from the detailed description to follow taken in conjugationwith the appended claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a represents the structure of monomeric PLB (left) and the aminoacid sequences of decoy peptides (right); the “L-shaped” structure ofmonomeric PLB is composed of cytoplasmic helix, a connecting short loop,and a transmembrane helix. Peptides were derived from the nine aminoacids that compose the connecting loop. In decoy peptides, Ser (S,serine) or Thr (T, threonine) residues were replaced with Glu (E,glutamic acid) to mimic phosphorylation. The replaced glutamic acidresidues are underlined. TAT (SEQ ID NO:11); RASTIEMPQ (SEQ ID NO:8);RAETIEMPQ (SEQ ID NO:1); RASEIEMPQ (SEQ ID NO:2).

FIG. 1b represents intracellular uptake of decoy peptides incardiomyocytes. The isolated cardiomyocytes were incubated with 1 μM ofpeptide solution for 1 hour at 37° C. Con, non-treated cell; ψPLB-SE,decoy peptides labeled with FITC; DIC, differential interferencecontrast image; FITC, FITC fluorescent image.

FIGS. 1c-1d represent the increase in PLB phosphorylation by the decoypeptides (FIG. 1c) and shortened decoy peptides (FIG. 1d). Isolatedadult cardiomyocytes were treated with 1 μM of indicated peptides for 45min, and then with 1 μM of PMA for 15 min. Western blots of cell lysateswere then probed with antibodies against phospho-PLB (S¹⁶), phospho-PLB(T¹⁷), total PLB, or GAPDH. The decoy peptides contain the followingsequences: ψPLB-SE, RAETIEMPQ (SEQ ID NO:1); ψPLB-SD, RADTIEMPQ (SEQ IDNO:3); ψPLB-TE, RASEIEMPQ (SEQ ID NO:2). The shortened decoy peptidescontain the following sequences: ψPLB-8-mer, AETIEMPQ (SEQ ID NO:4);ψPLB-7-mer, RAETIEM (SEQ ID NO:5); ψPLB-6-mer, RAETIE (SEQ ID NO:6);ψPLB-5-mer: RAETI (SEQ ID NO:7). PMA, phorbol 12-myristate 13-acetate.

FIGS. 2a-2d represent the increase in cardiac contractility by the decoypeptides. Isolated adult cardiomyocytes were treated with 1 μM ofindicated peptides for 45 min, and then with 1 μM of PMA for 15 min.Contractile parameters were then determined. FIGS. 2a and 2b showcontractile parameters. Peak cell shortening, percentage of shortenedcell length; −dL/dt, maximal rate of cell shortening; +dL/dt, maximalrate of cell relengthening. FIGS. 2c and 2d show average parameters ofthe transient Ca²⁺ properties determined with Fura2/AM. Baseline[Ca²⁺]_(i), baseline intracellular Ca²⁺ levels; Δ[Ca²⁺]_(i) (340/380),increase in intracellular Ca²⁺ levels in response to electric Stimuli;τ(ms), Ca²⁺ transient decay rate. Approximately 500 cells were chosenfor the contractility and Ca²⁺ transient measurements were taken fromeight individual hearts. #, P<0.05; *, P<0.01 compared to control; errorbars represent SD.

FIGS. 3a-3b represent the postischemic cardiac performance ex vivo. FIG.3a shows rat hearts which were Langendorff perfused, and subjected to 20min of no-flow to induce global ischemia, and then followed by 30 min ofreperfusion with co-administration of 1 μM TAT or ψPLB-SE. The changesin left ventricular developed pressure (LVDP) are shown. n=4. *, P<0.01compared to TAT; error bars represent SEM. FIG. 3b shows protein lysateswhich were prepared following reperfusion. Western blots probed withantibodies against phospho-PLB (S¹⁶), phosphor-PLB (T¹⁷), total PLB,Caspase 3, or GAPDH are shown.

DETAILED DESCRIPTION OF THIS INVENTION

In one aspect of this invention, there is provided a decoy peptide orpolypeptide consisting of a peptide sequence represented by thefollowing Formula I:X₁-Ala-X₂—X₃-Ile-Glu-X₄  (I)

wherein X₁ represents 0-50 amino acid residue(s), X₂ represents Ser,Glu, or Asp, X₃ represents Thr, Glu, or Asp and X₄ represents 0-50 aminoacid residue(s), with the proviso that X₂ is not Ser when X₃ is Thr;wherein the decoy peptide or polypeptide inhibits the PP1-mediateddephosphorylation of PLB by a competitive inhibition.

The present inventors have made intensive researches to a decoy peptideor polypeptide for treating diseases associated with PBL, in particular,caused by decreased SERCA2a activity by dephosphorylation levels of PLB.As a result, we have synthesized a decoy peptide which significantlyelevates phosphorylation levels of PLB. In addition, we have discoveredthat the decoy peptide increases contractile parameters in vitro andimproves left ventricular developed pressure ex vivo. That is to say, wehave found out that the decoy peptide or polypeptide providescardio-protective effects by restoring of SERCA2a activity and inotropiceffect of enhancing myocardial contractility and thus the decoy peptideor polypeptide would be used to treat the diseases associated with PLBby inhibiting dephosphorylation of PLB.

The decoy peptide or polypeptide of the present invention consisting ofa peptide sequence represented by the following Formula I:X₁—Ala-X₂—X₃-Ile-Glu-X₄ (I).

The decoy peptide or polypeptide designed to mimic phosphorylated PLBinhibits PP1-mediated dephosphorylation of PLB and significantlyelevates phosphorylation levels of PLB and increases contractileparameters, thereby significantly improving left ventricular developedpressure. The decoy peptide or polypeptide provides an alternativemodality for the restoration of SERCA2a activity in failing hearts.

In the decoy peptide or polypeptide of the present invention, thesequence “Ala-X₂—X₃-Ile-Glu” is necessary for its actions and functions.The X₁ and X₄ residues may have lots of variations. In this regard, thepresent invention encompasses any peptide or polypeptide comprising thesequence “Ala-X₂—X₃-Ile-Glu” so long as it retains functions oractivities as a decoy to PP1.

The term used herein “peptide” means a linear molecule formed by linkingamino acid residues via peptide bonds. The term used herein“polypeptide” means any polymer of (same or different) amino acidsjoined via peptide bonds.

The term used herein “decoy peptide or polypeptide” in conjunction withPLB is the designed peptide or polypeptide comprising a peptide sequencethat mimics a connecting short loop of phosphorylated PLB and is capableof binding to PP1 in a competitive manner, thereby blocking the actionof PP1.

The term used herein “PP1-mediated dephosphorylation” means thedephosphorylation of PLB by PP1.

The term used herein “competitive inhibition” with reference to decoypeptide (or polypeptide) means inhibition of dephosphorylation bybinding competitively to PP1 to form the decoy peptide orpolypeptide-PP1 complex. The decoy peptide or polypeptide binds to PP1in such a way that it competes with phosphorylation sites of PLB forbinding to PP1.

In certain embodiments, the decoy peptide or polypeptide of the presentinvention is cytosolic peptide or polypeptide. That is to say, inFormula I, X₁ and X₄ do not contain an amino acid domain which wouldpreclude the decoy peptide or polypeptide from existing in the cytosol.For example, the amino acid domain includes membrane-spanning domainsand organelle-targeting domains, but not limited to. In this regard, X₁and X₄ encompass any amino acid residue(s) so long as they allow thedecoy peptide or polypeptide to locate in the cytosol.

The decoy peptide or polypeptide may be in any length as long as itinhibits PP1-mediated dephosphorylation of PLB. For example, the decoypeptide or polypeptide may be in length of 5-100, 5-80, 5-60, 5-40,5-30, 5-20, 5-15, 5-9, or 6-9 amino acids.

In certain embodiments of Formula I, X₁ represents 0-40, 0-30, 0-20,0-10, 0-3, or 0-1 amino acid residue(s).

In Formula I, any amino acid(s) is located at X₁. In certainembodiments, X₁ consists of an amino acid sequence spanning 0-50, 0-40,0-30, 0-20, 0-10, 0-3, or 0-1 amino acid residue(s) in the N-terminaldirection of the amino acid position 15 of the amino acid sequence ofPLB (see SEQ ID NO:10). In other embodiments, X₁ consists of 1 aminoacid residue in the N-terminal direction of the amino acid position 15of the amino acid sequence of PLB (see SEQ ID NO:10).

In certain embodiments of Formula I, X₄ represents 0-40, 0-30, 0-20,0-10, or 0-3 amino acid residue(s).

In Formula I, any amino acid(s) is located at X₄. In certainembodiments, X₄ comprises an amino acid sequence spanning 0-50, 0-40,0-30, 0-20, 0-10, or 0-3 amino acid residue(s) in the C-terminaldirection of the amino acid position 19 of the amino acid sequence ofPLB (see SEQ ID NO:10). In other embodiments, X₄ consists of an aminoacid sequence spanning 0 or 3 amino acid residue(s) in the N-terminaldirection of the amino acid position 19 of the amino acid sequence ofPLB (see SEQ ID NO:10). In other embodiments, X₄ consists of an aminoacid sequence spanning 3 amino acid residues in the N-terminal directionof the amino acid position 19 of the amino acid sequence of PLB (see SEQID NO:10).

In other embodiments, wherein X₁ is Arg.

In other embodiments, wherein X₄ is Met, Met-Pro, or Met-Pro-Gln.

The Ser residue at the amino acid position 16 (Ser¹⁶) and Thr residue atthe amino acid position 17 (Thr¹⁷) of the amino acid sequence of PLB(see SEQ ID NO:10) are the phosphorylation sites located within theflexible loop region (the amino acid position 14-22 of the amino acidsequence of PLB (see SEQ ID NO:10)) of PLB. In Formula I, X₂ and X₃represents the amino acid position occupied by the Ser¹⁶ and Thr¹⁷ inPLB, respectively.

In the present invention, the decoy peptide or polypeptide is designedby substituting the Ser¹⁶ and/or Thr¹⁷ residue(s) with a Glu or Aspresidue. The decoy peptide or polypeptide comprising a Glu or Aspresidue at X₂ and/or X₃ of Formula I is similar to phosphorylated PLB,thereby competing with the phosphorylation sites of PLB for binding toPP1.

In other embodiments, X₂ is Glu or Asp and X₃ is Thr, Glu, or Asp. Inother embodiments, X₂ is Glu or Asp and X₃ is Thr.

In particular embodiments, the decoy peptide or polypeptide consists ofthe amino acid sequence selected from amino acid sequences as set forthin SEQ ID NOs:1-6. In particular embodiments, the decoy peptide orpolypeptide consists of the amino acid sequence selected from amino acidsequences as set forth in SEQ ID NOs:1 and 3-6. In particularembodiments, the decoy peptide or polypeptide consists of the amino acidsequence selected from amino acid sequences as set forth in SEQ IDNOs:1, 3 and 6.

The sequences SEQ ID NOs:1-6 are as follows:

The sequence SEQ ID NO:1 is Arg-Ala-Glu-Thr-Ile-Glu-Met-Pro-Gln.

The sequence SEQ ID NO:2 is Arg-Ala-Ser-Glu-Ile-Glu-Met-Pro-Gln.

The sequence SEQ ID NO:3 is Arg-Ala-Asp-Thr-Ile-Glu-Met-Pro-Gln.

The sequence SEQ ID NO:4 is Ala-Glu-Thr-Ile-Glu-Met-Pro-Gln.

The sequence SEQ ID NO:5 is Arg-Ala-Glu-Thr-Ile-Glu-Met.

The sequence SEQ ID NO:6 is Arg-Ala-Glu-Thr-Ile-Glu.

The decoy peptide or polypeptide of the present invention may includepeptide or polypeptide in which one or more of amino acids have sidechain modification. Examples of side chain modifications includemodifications of amino groups such as by reductive alkylation;amidination with methylacetimidate; acylation with acetic anhydride;carbamolyation of amino groups with cyanate; trinitrobenzylation ofamino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS);acylation of amino groups with succinic anhydride and tetrahydrophthalicanhydride; and pyridoxylation of lysine with pyridoxal-5-phosphatefollowed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal. The carboxyl group may bemodified by carbodiimide activation via O-acylisourea formation followedby subsequent derivitisation, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylationwith iodoacetic acid or iodoacetamide; performic acid oxidation tocysteic acid; formation of a mixed disulphides with other thiolcompounds; reaction with maleimide, maleic anhydride or othersubstituted maleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-chloromercuri-4-nitrophenol and othermercurials; carbamoylation with cyanate at alkaline pH. Any modificationof cysteine residues must not affect the ability of the peptide to formthe necessary disulphide bonds. It is also possible to replace thesulphydryl group of cysteine with selenium equivalents such that thepeptide forms a diselenium bond in place of one or more of thedisulphide bonds.

Tryptophan residues may be modified by, for example, oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residueson the other hand, may be altered by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carbethoxylation with diethylpyrocarbonate.

Proline residue may be modified by, for example, hydroxylation in the4-position.

The decoy peptide or polypeptide of the present invention possesses muchhigher stability by modifications. For example, the decoy peptide orpolypeptide has at least one amino acid residue protected with acetylgroup, fluorenyl methoxy carbonyl group, formyl group, palmitoyl group,myristyl group, stearyl group or polyethylene glycol. In certainembodiments, the decoy peptide or polypeptide has at least one aminoacid residue protected with acetyl group.

The term used herein “stability” refers to in vivo stability and storagestability (e.g., storage stability at room temperature) as well. Theprotection group described above protects the peptides from the attackof protease in vivo.

In other embodiments, the decoy peptide or polypeptide is further linkedto a cell membrane-permeable peptide. In other embodiments, either theN-terminal and/or C-terminal of the decoy peptide or polypeptide isfurther linked to a cell membrane-permeable peptide.

For the decoy peptide or polypeptide of the present invention to betransferred into cardiaomyocytes, it may contain the cellmembrane-permeable peptide. The term used herein “cellmembrane-permeable peptide” means a peptide necessarily required tointroduce a specific peptide (or protein) into a cell. Usually, itconsists of 5-50 or more amino acid sequences.

The cell membrane-permeable peptide is a peptide capable of passingthrough the phospholipid bilayer of the cell membrane as it is. Forexample, it includes a Tat-derived peptide, a signal peptide (e.g., acell-penetrating peptide), an arginine-rich peptide, a transportan, oran amphiphipathic peptide carrier, but not limited to (Morris, M. C. etal., Nature Biotechnol. 19: 1173-1176 (2001); Dupont, A. J. andProchiantz, A., CRC Handbook on Cell Penetrating Peptides, Langel,Editor, CRC Press (2002); Chaloin, L. et al., Biochemistry 36(37):11179-87 (1997); and Lundberg, P. and Langel, U., J. Mol. Recognit.16(5): 227-233 (2003)). In addition to these naturally occurringpeptides, various antennapedia-based peptides capable of crossing thecell membrane are known, including retroinverso and D-isomer peptides(Brugidou, 3. et al., Biochem Biophys Res Commun. 214(2): 685-93 (1995);Derossi, D. et al., Trends Cell Biol. 8: 84-87 (1998)).

In certain embodiments, the TAT peptide (Tat-derived peptide) may beused as the cell membrane-permeable peptide.

The Tat protein, which originates from human immunodeficiency virus(HIV), consists of 86 amino acids and has cysteine-rich, basic andintegrin-binding domains as major protein domains. Although the TATpeptide has a cell membrane-penetrating property only with theYGRKKRRQRRR (SEQ ID NO:11) (i.e., the 48^(th) to 60^(th) amino acids ofTat protein) sequence, it is known that a more efficient penetration ispossible when it has a branched structure including several copies ofthe RKKRRQRRR sequence (Tung, C. H. et al., Bioorg. Med. Chem. 10:3609-3614 (2002)). The various Tat peptides having cellmembrane-penetrating ability are described in Schwarze, S. R. et al.,Science 285: 1569-1572 (1999). In certain embodiments, the TAT peptidecomprises the amino acid sequence as set forth in SEQ ID NO:11.

In addition, the decoy peptide or polypeptide of the present inventionmay further comprise the fusion protein for convenient purification,which includes but not limited to, with glutathione S-transferase(Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA),or 6× His (hexahistidine; Quiagen, USA). In certain embodiments, thefusion protein may be purified by affinity chromatography. For example,elution buffer containing glutathione is employed for fusion proteinswith glutathione S-transferase and Ni-NTA His-binding resin (Novagen,USA) is employed for fusion proteins with 6× His to purify the fusionprotein of interest in a rapid and feasible manner.

In other embodiments, PLB is derived from human and its amino acidsequences are disclosed in NCBI (National Center for BiotechnologyInformation). Examples of Accession Numbers of the human PLB amino acidsequences in NCBI are AAA60109.1, AAA60083.1 and AAD55950.1.

According to the present invention, the term used herein “the decoypeptide or polypeptide” is intended to include functional equivalents ofthe decoy peptide or polypeptide. As used herein, the term “functionalequivalent” refers to amino acid sequence variants (for example,variations at amino acid residues surrounding the necessary sequenceAla-X₂—X₃-Ile-Glu) having amino acid substitutions, additions ordeletions in some of the amino acid sequence of the decoy peptide andpolypeptide while simultaneously having similar or improved biologicallyactivity when compared to the decoy peptide and polypeptide. The aminoacid substitutions may be conservative substitutions. Examples of theconservative substitutions of naturally occurring amino acids includealiphatic amino acids (Gly, Ala, and Pro), hydrophobic amino acids (Ile,Leu, and Val), aromatic amino acids (Phe, Tyr, and Trp), acidic aminoacids (Asp, and Glu), basic amino acids (His, Lys, Arg, Gln, and Asn),and sulfur-containing amino acids (Cys, and Met). The deletions of aminoacids are located in a region which is not involved directly in theactivity of the decoy peptides and polypeptides.

According to the present invention, the amino acid sequences of thedecoy peptide and polypeptide available to the present invention areintended to include peptide sequences having substantial identity to thedecoy peptide sequences. The term “substantial identity” as used hereinmeans that the two amino acid sequences, when optimally aligned, such asby the program BLAST, GAP, or BESTFIT, or by visual inspection, share atleast about 60%, 70%, 80%, 85%, 90%, or 95% sequence identity orsequence similarity. Methods of alignment of sequences for comparisonare well-known in the art. Various programs and alignment algorithms aredescribed in: Smith and Waterman, Adv. Appl. Math. 2:482 (1981),Needleman and Wunsch, J. Mol. Bio. 48:443 (1970), Pearson and Lipman,Methods in Mol. Biol. 24: 307-31 (1988), Higgins and Sharp, Gene73:237-44 (1988), Higgins and Sharp, CABIOS 5:151-3 (1989), Corpet etal., Nuc. Acids Res. 16:10881-90 (1988), Huang et al., Comp. Appl.BioSci. 8:155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24:307-31(1994) presents a detailed consideration of sequence alignment methodsand homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-10 (1990)) is available from several sources,including the NCBI and on the internet, for use in connection with thesequence analysis programs blastp, blasm, blastx, tblastn and tblastx.It can be accessed at http://www.ncbi.nlm.nih.gov/BLAST/. A descriptionof how to determine sequence identity using this program is available athttp://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

In another aspect of this invention, there is provided a pharmaceuticalcomposition for preventing or treating a disease associated with PLB,comprising (a) a pharmaceutically effective amount of the decoy peptideor polypeptide of any one of the decoy peptides or polypeptides of thepresent invention; and (b) a pharmaceutically acceptable carrier.

The term used herein “preventing” with reference to a disease associatedwith PLB refers to the complete prevention of a disease associated withPLB, the prevention of occurrence of symptoms in a subject with thedisease or the prevention of recurrence of symptoms in a subject withthe disease.

The term used herein “treating” with reference to a disease associatedwith PLB refers to the partial or total elimination of symptoms ordecrease in severity of symptoms of a disease associated with PLB in thesubject.

The term used herein “pharmaceutically effective amount” with referenceto a disease associated with PLB means a sufficient dose in the subjectto which it is administered, to prevent or treat the symptoms,conditions, or diseases associated with PLB.

The term used herein “subject” is intended to encompass human, non-humanmammal, or animal. Non-human mammals include livestock animals andcompanion animals, such as cattle, sheep, goats, equines, swine, dogs,and cats.

According to the present invention, the pharmaceutical compositioncomprising the decoy peptide or polypeptide as an active ingredientwould be used to prevent or treat a disease associated with PLB.

The contractility of cardiomyocytes is directly regulated byintracellular Ca²⁺ cycling, and SERCA2a plays a crucial role in Ca²⁺handling in cardiomyocytes. PLB is an endogenous inhibitor of SERCA2aand its inhibitory activity is enhanced via dephosphorylation by PP1.

In the present invention, the decoy peptide significantly elevatesphosphorylation levels of PLB (see FIGS. 1c and 1d). In addition, thedecoy peptide increases contractile parameters in vitro (see FIGS.2a-2d) and improves left ventricular developed pressure ex vivo (seeFIGS. 3a and 3b). That is to say, the decoy peptide or polypeptideprovides cardio-protective effects by restoring of SERCA2a activity andinotropic effect of enhancing myocardial contractility and thus thedecoy peptide or polypeptide would be used to prevent or treat thediseases associated with PLB by inhibiting PP1-mediateddephosphorylation of PLB.

In certain embodiments, the disease associated with PLB is a heartdisease. In certain embodiments, the heart disease is heart failure,ischemia, arrhythmia, myocardial infarction, congestive heart failure,transplant rejection, abnormal heart contractility, or abnormal Ca²⁺metabolism. In certain embodiments, the heart disease is heart failureor ischemia. In particular embodiments, the heart disease is heartfailure.

The term used herein “heart failure” means a clinical symptom in whichthe stroke volume of the heart decreases below a normal value and theheart fails to supply enough blood to peripheral tissues. In otherwords, heart failure means the state in which the ability of the heartto pump blood is decreased due to various causes or enough blood cannotbe supplied to the body even when the heart beats normally.

In other embodiments, the heart failure is induced by cardiachypertrophy, coronary arteriosclerosis, myocardial infarction, valvularheart disease, hypertension, or cardiomyopathy.

In other embodiments, the pharmaceutical composition is an inotropicpharmaceutical composition.

According to the present invention, the pharmaceutical composition maycontain pharmaceutically acceptable carriers. The pharmaceuticallyacceptable carrier may be conventional one for formulation, includinglactose, dextrose, sucrose, sorbitol, mannitol, starch, rubber arable,potassium phosphate, arginate, gelatin, potassium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water,syrups, methyl cellulose, methylhydroxy benzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils, but not limitedto. The pharmaceutical composition according to the present inventionmay further include a lubricant, a humectant, a sweetener, a flavoringagent, an emulsifier, a suspending agent, and a preservative. Details ofsuitable pharmaceutically acceptable carriers and formulations can befound in Remington's Pharmaceutical Sciences (19th ed., 1995), which isincorporated herein by reference.

The pharmaceutical composition according to the present invention may beorally or parentally administered. When the pharmaceutical compositionof the present invention is administered parenterally, it can be done byintravenous, subcutaneous, intramuscular, abdominal or transdermaladministration.

A suitable dosage amount of the pharmaceutical composition of thepresent invention may vary depending on pharmaceutical formulationmethods, administration methods, the patient's age, body weight, sex,pathogenic state, diet, administration time, administration route, anexcretion rate and sensitivity for a used pharmaceutical composition,and physicians of ordinary skill in the art can determine an effectiveamount of the pharmaceutical composition for desired treatment.Generally, suitable dosage unit for human host is to administer with thepharmaceutical composition in 0.0001-100 mg/kg (body weight).

According to the conventional techniques known to those skilled in theart, the pharmaceutical composition may be formulated withpharmaceutically acceptable carrier and/or vehicle as described above,finally providing several forms including a unit dose form and amulti-dose form. Formulation may be oil or aqueous media, resuspensionor emulsion, extract, powder, granule, tablet and capsule and furthercomprise dispersant or stabilizer.

In another aspect of this invention, there is provided a method forpreparing a decoy peptide or polypeptide of PLB capable of inhibitingPP1-mediated dephosphorylation of PLB by a competitive inhibition,comprising (a) designing the decoy peptide or polypeptide of PLB bysubstituting a Ser residue at the amino acid position 16 and/or a Thrresidue at the amino acid position 17 of the amino acid sequence of PLB(SEQ ID NO:10) with a Glu or Asp residue and selecting 0-95 surroundingamino acid residue(s) around the amino acid position 15-19 of the aminoacid sequence of PLB (SEQ ID NO:10), such that the decoy peptide orpolypeptide designed consists of a peptide sequence represented by thefollowing Formula I: X₁-Ala-X₂—X₃-Ile-Glu-X₄ (I) wherein X₁ represents0-50 amino acid residue(s), X₂ represents Ser, Glu, or Asp, X₃represents Thr, Glu, or Asp and X₄ represents 0-50 amino acidresidue(s), with the proviso that X₂ is not Ser when X₃ is Thr; and (b)preparing the decoy peptide or polypeptide designed in the step (a).

The present invention will be described in more detail as follows:

In the first step, the decoy peptide or polypeptide of PLB is designedby substituting a Ser residue at the amino acid position 16 and/or a Thrresidue at the amino acid position 17 of the amino acid sequence of PLB(SEQ ID NO:10) with a Glu or Asp residue and selecting 0-95 surroundingamino acid residue(s) around the amino acid position 15-19 of the aminoacid sequence of PLB (see SEQ ID NO:10), such that the decoy peptide orpolypeptide designed consists of a peptide sequence represented by thefollowing Formula I: X₁—Ala-X₂—X₃-Ile-Glu-X₄ (I) wherein X₁ represents0-50 amino acid residue(s), X₂ represents Ser, Glu, or Asp, X₃represents Thr, Glu, or Asp and X₄ represents 0-50 amino acidresidue(s), with the proviso that X₂ is not Ser when X₃ is Thr.

In the decoy peptide or polypeptide of the present invention, the aminoacid residues at the amino acid position 15, 18, and 19 of the aminoacid sequence of PLB are Ala, Ile, and Glu (see SEQ ID NO:10).

According to the present invention, “Ala-X₂—X₃-Ile-Glu” of the decoypeptide or polypeptide sequence is necessary for its actions andfunctions. The surrounding amino acid residue(s) may have lots ofvariations. In this regard, the decoy peptide or polypeptide encompassesany surrounding amino acid residue(s) so long as it retains functions oractivities as a decoy to PP1.

The surrounding amino acid residue(s) may be in any length as long as itinhibits PP1-mediated dephosphorylation of PLB. For example, thesurrounding amino acid residue(s) may be in length of 0-95, 0-75, 0-55,0-35, 0-15, 0-10, 0-4, or 1-4 amino acid.

In other embodiments, the surrounding amino acid residue(s) consists of(i) an amino acid sequence spanning 0-50, 0-40, 0-30, 0-20, 0-10, 0-3,or 0-1 amino acid residue(s) in the N-terminal direction of the aminoacid position 15 and (ii) an amino acid sequence spanning 0-50, 0-40,0-30, 0-20, 0-10, or 0-3 amino acid residue(s) in the C-terminaldirection of the amino acid position 19 of the amino acid sequence ofPLB (SEQ ID NO:10). In other embodiments, the amino acid residue in theN-terminal direction of amino acid position 15 is 1 amino acid residueand the amino acid residue(s) in the C-terminal direction of amino acidposition 19 is 0 or 3 amino acid residue(s).

In other embodiment, the amino acid residue in the N-terminal directionof the amino acid position 15 is Arg. In other embodiments, the aminoacid residue(s) in the C-terminal direction of the amino acid position19 is is Met, Met-Pro, or Met-Pro-Gln.

In the present invention, the decoy peptide or polypeptide substitutedthe Ser¹⁶ or Thr¹⁷ residue(s) with a Glu or Asp residue increase thephosphorylation of PLB (see FIGS. 1c and 1d). Therefore, even if theonly one of Ser¹⁶ and Thr¹⁷ residues is substituted with Glu or Aspresidue, the decoy peptide or polypeptide could inhibit PP1-mediateddephosphorylation of PLB.

In certain embodiments, the decoy peptide or polypeptide is designed bysubstituting the Ser residue or both the Ser and the Thr residues with aGlu or Asp residue. In certain embodiments, the decoy peptide orpolypeptide is designed by substituting only the Ser residue with a Gluor Asp residue.

In particular embodiments, the decoy peptide or polypeptide is designedsuch that it consists of the amino acid sequence selected from aminoacid sequences as set forth in SEQ ID NOs:1-6. In particularembodiments, the decoy peptide or polypeptide is designed such that itconsists of the amino acid sequence selected from amino acid sequencesas set forth in SEQ ID NOs:1 and 3-6. In particular embodiments, thedecoy peptide or polypeptide is designed such that it consists of theamino acid sequence selected from amino acid sequences as set forth inSEQ ID NOs:1, 3 and 6.

Following the step (a), the decoy peptide or polypeptide designed in thestep (a) is prepared.

The decoy peptide or polypeptide of the present invention may beprepared according to recombinant DNA technologies or the solid-phasesynthesis technique commonly employed in the art (Merrifield, R. B., J.Am, Chem. Soc., 85: 2149-2154 (1963), Kaiser, E., Colescot, R. L.,Bossinger, C. D., Cook, P. I., Anal. Biochem., 34: 595-598 (1970)). Theamino acids with α-amino and side-chain groups protected are attached toa resin. Then, after removing the α-amino protecting groups, the aminoacids are successively coupled to obtain an intermediate.

In still another aspect of this invention, there is provided a methodfor preventing or treating a disease associated with PLB, comprisingadministering to a subject in need thereof (a) a pharmaceuticallyeffective amount of the decoy peptide or polypeptide of any one of thedecoy peptides or polypeptides of the present invention; and (b) apharmaceutically acceptable carrier.

In certain embodiments, the disease associated with PLB is a heartdisease. In certain embodiments, the heart disease is heart failure,ischemia, arrhythmia, myocardial infarction, congestive heart failure,transplant rejection, abnormal heart contractility, or abnormal Ca²⁺metabolism. In certain embodiments, the heart disease is heart failureor ischemia. In particular embodiments, the heart disease is heartfailure.

In other embodiments, the heart failure is induced by cardiachypertrophy, coronary arteriosclerosis, myocardial infarction, valvularheart disease, hypertension, or cardiomyopathy.

The features and advantages of this invention will be summarized asfollows:

(a) The present invention provides a decoy peptide or polypeptidecapable of inhibiting PP1-mediated dephosphorylation of PLB by acompetitive inhibition and a pharmaceutical composition for preventingor treating a disease associated with PLB comprising the decoy peptideor polypeptide of the present invention as an active ingredient.

(b) The present invention provides a method for preparing the decoypeptide or polypeptide of PLB and preventing or treating a diseaseassociated with PLB.

(c) It is noteworthy that the decoy peptide or polypeptide of thepresent invention significantly elevates phosphorylation levels of PLBby inhibiting PP1-mediated dephosphorylation. In addition, the decoypeptide or polypeptide provides cardio-protective effects by restoringof SERCA2a activity and inotropic effect of enhancing myocardialcontractility.

(d) The present invention will contribute greatly to the prevention ortreatment of diseases associated with PLB.

The present invention will now be described in further detail byexamples. It would be obvious to those skilled in the art that theseexamples are intended to be more concretely illustrative and the scopeof the present invention as set forth in the appended claims is notlimited to or by the examples.

EXAMPLES

Materials and Methods

Decoy Peptides

The decoy peptides were derived from the PLB protein sequence (SEQ IDNO:10) surrounding the phosphorylation sites Ser¹⁶ and Thr¹⁷:RAS¹⁶T¹⁷IEMPQ. The peptides were conjugated via a cystein-cystein bondat their N termini to the cell penetrating peptide TAT (YGRKKRRQRRR (SEQID NO:11)) to facilitate uptake into cells. The peptides used in thisinvention were as follows: ψPLB-wt, RASTIEMPQ (SEQ ID NO:8); ψPLB-SE,RAETIEMPQ (SEQ ID NO:1); ψPLB-TE, RASEIEMPQ (SEQ ID NO:2); ψPLB-SD,RADTIEMPQ (SEQ ID NO:3). In addition, shortened ψPLB-SE peptides werealso tested: 8-mer, AETIEMPQ (SEQ ID NO:4); 7-mer, RAETIEM (SEQ IDNO:5); 6-mer, RAETIE (SEQ ID NO:6); 5-mer, RAETI (SEQ ID NO:7). All ofthe peptides were synthesized and modified by AnyGen (Gwangju, Korea)and dissolved in distilled water to a stock concentration of 1 mM.Peptides were >95% pure. The decoy peptides were treated for 1 h at afinal concentration of 1 μM. Cell viability and morphology were notsignificantly affected by the peptides

Isolation of Adult Rat Ventricular Myocytes

Ventricular myocytes were isolated from SD rat hearts as previouslydescribed [33] with minor modifications. Male rats of 8-12 weeks of age(250-320 g) were used. In brief, rats were anesthetized by theinhalation of isofluran (0.5%) for 5 min. The heart was quickly removedfrom the chest and the aorta was retrogradely perfused at 37° C. for 3min with calcium-free Tyrode buffer (137 mM NaCl, 5.4 mM KCl, 1 mMMgCl₂, 10 mM glucose, 10 mM HEPES [pH 7.4], 10 mM 2, 3-butanedionemonoxime, and 5 mM taurine) gassed with 100% O₂. The enzymatic digestionwas then initiated by adding collagenase type B (0.35 U/ml; Roche) andhyaluronidase (0.1 mg/ml; Worthington) to the perfusion solution. Whenthe heart became swollen after 10 min of digestion, the left ventriclewas quickly removed, cut into several chunks, and further digested in ashaker (60-70 rpm) for 10 min at 37° C. in the same enzyme solution. Thesupernatant containing the dispersed myocytes was then filtered througha cell strainer (100 μm in pore size, BD Falcon) and gently centrifugedat 500 rpm for 1 min. Extracellular Ca²⁺ was incrementally added back toa concentration of 1.25 mM over a span of 30 min to avoid the Ca²⁺paradox. This procedure usually yielded ≥80% viable rod-shapedventricular myocytes with clear sarcomere striations. Myocytes withobvious sarcolemmal blebs or spontaneous contractions were discarded.

Fluorescence Microscopy

To confirm cellular uptake of the peptides, ψPLB-SE was labeled withFITC. The isolated cardiomyocytes were plated onto a laminin-coatedglass plate and cultured in modified Eagle's Medium (MEM) with Hanks'Balanced Salt solution, supplemented with 2 mM L-carnitine, 5 mMcreatine and 5 mM taurine, and 100 IU/ml penicillin. The cells wereexposed to 1 μM FITC-labeled ψPLB-SE for 1 h, and then washed twice withTyrode solution. Fluorescence images were visualized using a Leica DMRBEmicroscope (LabCommerce Inc.) equipped with a 63× (1.4 NA) oil objectiveand fluorescein FITC-optimized filter sets (OmegaR Optical Inc.). Imageswere acquired using a CoolSNAP TMfx CCD camera (Photometrics) andanalyzed with Metamorph imaging software (Universal Imaging Co.).

Western Blot Analysis.

Heart lysates (50 μg) in SDS sample buffer were run on a SDS-PAGE geland then transferred to a PVDF membrane (Bio-Rad). The membrane wasblocked with a 5% skim milk solution and then incubated overnight withantibodies directed against PLB (Affinity Bioreagents), phospho-PLB(Ser¹⁶, Cell Signaling), phospho-PLB (Thr¹⁷, Badrilla), SERCA2a (SantaCruz), Caspase 3 (Cell Signaling), or GAPDH (Santa Cruz). The membraneswere then incubated with a secondary antibody conjugated to horseradishperoxidase (Jackson Immuno Research) and developed using the WesternLighting chemiluminescence reagent (Perkin Elmer).

Cell Contractility and Intracellular Ca²⁺ Transient Measurements

The mechanical properties of ventricular myocytes were assessed using avideo-based edge detection system (IonOptix), as previously described[34]. In brief, laminin-coated coverslips with attached cells wereplaced in a chamber mounted on the stage of an inverted microscope(Nikon Eclipse TE-100F) and perfused (about 1 ml/min at 37° C.) withTyrode buffer (137 mM NaCl, 5.4 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mMglucose, and 10 mM HEPES [pH 7.4]). The cells were field-stimulated at afrequency of 3 Hz (30 V) using a STIM-AT stimulator/thermostat placed ona HLD-CS culture chamber/stim holder (Cell Micro Controls). The myocyteof interest was displayed on a computer monitor using an IonOptix MyoCamcamera, which rapidly scanned the image area every 8.3 ms, so that theamplitude and velocity of shortening or relengthening were recorded withfidelity. Changes in cell length during shortening and relengtheningwere captured and analyzed using soft edge software (IonOptix). Thecardiomyocytes were loaded with 0.5 μM Fura2-AM (Molecular Probes), aCa²⁺-sensitive indicator, for 15 min at 37° C. Fluorescence emissionswere recorded simultaneously with the contractility measurements usingMyocyte calcium and contractility recording system (IonOptix).Cardiomyocytes were exposed to light emitted by a 75 W halogen lampthrough either a 340 or 380 nm filter while being field-stimulated asdescribed above. Fluorescence emissions were detected between 480 and520 nm by a photomultiplier tube after initial illumination at 340 nmfor 0.5 ms and then at 380 nm for the duration of the recordingprotocol. The 340 nm excitation scan was then repeated at the end of theprotocol, and qualitative changes in the intracellular Ca²⁺concentration were inferred from the ratio of the Fura fluorescenceintensity at both wavelengths.

Isolated Perfused Heart Experiments

Rats were anesthetized by inhalation of isofluran (0.5%) for 5 min. Theheart was quickly removed and placed in cold oxygenated Tyrode solution(137 mM NaCl, 5.4 mM KCl, 1 mM MgCl₂, 10 mM glucose, 1 mM CaCl₂, 10 mMHEPES [pH 7.4], 100% O₂). The aorta was cannulated and perfused by theLangendorff method with oxygenated Tyrode solution at constant pressure(65 mmHg, temperature 37±0.2° C.). A latex water-filled balloon wasinserted into the left ventricular chamber and connected to a pressuretransducer (AD Instruments) for continuous measurement of heartperformance. Heart rate, left ventricular developed pressure (LVDP), andthe first derivatives of LV pressure (LV +dP/dt max and LV −dP/dt max)were all recorded using PowerLab Chart Systems (AD Instruments). Theballoon volume was adjusted in 250-300 μl to result in a measured enddiastolic pressure in the range of 6-10 mmHg. After a 30 minstabilization period, the hearts were subjected to 20 min of no-flow toinduce global ischemia, followed by 30 min of reperfusion. Perfusion ofdecoy peptides was performed at a final concentration of 1 μM.

Statistics

Where appropriate, the data are expressed as means±SDs. Comparisons ofgroup means were made by using either a Student's t-test or a one-wayANOVA with Bonferroni correction (Statview version 5.0, SAS). A P-valueof <0.05 was considered to be statistically significant.

Results

Decoy Peptide

PLB is composed of a cytoplasmic helix at the N-terminus, atransmembrane helix at the C-terminus, and a flexible loop that connectsthe two helices (FIG. 1a, left). The phosphorylation sites Ser¹⁶ andThr¹⁷ reside in the connecting loop region. We synthesized a 9-merpeptide (RASTIEMPQ (SEQ ID NO:8)) that exactly matched the nine aminoacid sequence of the loop region and coupled it to a cell-permeablepeptide TAT through a disulfide bond to yield ψPLB-wt. In addition, wegenerated similar TAT-coupled peptides, in which the Ser or Thr residuecorresponding to Ser¹⁶ or Thr¹⁷ of PLB were individually replaced by Gluto yield ψPLB-SE or ψPLB-TE, respectively (FIG. 1a, right). Thesepeptides were labeled with FITC and added to the cultured cardiomyocytesisolated from rat left ventricles. Observation under a fluorescentmicroscope revealed an efficient intracellular uptake of the peptides invirtually all of the cardiomyocytes (FIG. 1b). This experiment ensuredthat the cardiomyocytes efficiently took up the peptides, and soun-labeled peptides were then used for the rest of the experiments.

It is known that the treatment of cardiomyocytes with PMA (phorbol12-myristate 13-acetate) significantly reduces phosphorylation of PLBthrough activation of PKCα. Pre-treatment with ψPLB-SE, but not withψPLB-wt, significantly blocked the PMA-induced dephosphorylation of PLBat both Ser¹⁶ and Thr¹⁷. ψPLB-TE also blocked dephosphorylation but wasless effective than ψPLB-SE (FIG. 1c). The elevated phosphorylationlevels of PLB seen after ψPLB-SE treatment were slightly butsignificantly higher than the basal levels. Considering that PP1 is theonly protein phosphatase known to dephosphorylate PLB, these datademonstrated that ψPLB-SE served as an effective decoy peptide tocompetitively inhibit PP1-mediated dephosphorylation of PLB.

ψPLB-SD, in which Ser¹⁶ was replaced with Asp, was as effective asψPLB-SE in elevating the phosphorylation levels of PLB (FIG. 1c). Wesynthesized shortened ψPLB-SE peptides consisting of AETIEMPQ (SEQ IDNO:4) (ψPLB-8-mer), RAETIEM (SEQ ID NO:5) (ψPLB-7-mer), RAETIE (SEQ IDNO:6) (ψPLB-6-mer), and RAETI (SEQ ID NO:7) (ψPLB-5-mer). Among thesepeptides, only ψPLB-5-mer was ineffective in elevating thephosphorylation levels of PLB (FIG. 1d). Therefore, it appeared thatASTIE (SEQ ID NO:9) is the minimal amino acid sequence required, andthat the Ser residue can be replaced by either Glu or Asp to create aneffective decoy peptide for PP1.

ψPLB-SE Increases Cardiomyocyte Contractility

We next tested whether ψPLB-SE affected cardiomyocyte contractility.Isolated adult cardiomyocytes were pre-treated with the peptides andthen treated with PMA. As indicated by the reduced contractileparameters including peak shortening, and the rates of contraction andrelaxation, contractility was significantly reduced by PMA. WhileψPLB-wt and ψPLB-TE elicited no or only marginal effects oncontractility, ψPLB-SE completely prevented the PMA-induced reduction incontractility (FIG. 2a). PMA also evoked a significant reduction in theCa²⁺ transient amplitude and a significant increase in the time constantof the Ca²⁺ transient decay (T). These PMA-induced defects werecompletely reversed by pre-treatment of ψPLB-SE, while the effects ofψPLB-wt and ψPLB-TE on intracellular Ca²⁺ handling were negligible ormarginal (FIG. 2b). These data indicated that the restoredphosphorylation levels of endogenous PLB by pre-treatment of ψPLB-SEyielded a normalized contractility. Both ψPLB-SD and ψPLB-6-mer were aseffective as ψPLB-SE in the preservation of contractility and Ca²⁺handling (FIGS. 2c and 2d).

ψPLB-SE Improves Functional Recovery after Ischemia/Reperfusion Ex Vivo

We further examined the benefits of ψPLB-SE during ischemia/reperfusion(I/R) ex vivo. Rat hearts were Langendorff-perfused, subjected to 20 minof no-flow to induce global ischemia, and then 30 min of reperfusion.The developed pressure of left ventricles was significantly lowered(37-44 mmHg vs. 80-100 mmHg at pre-ischemia) by I/R. At the time ofreperfusion, ψPLB-SE or a control peptide TAT was added to thereperfusion solutions. While TAT had no effects, ψPLB-SE significantlyelevated the developed pressure (73-78 mmHg) (FIG. 3a). At the end ofthe experiment, lysates of the hearts were prepared and subjected toWestern blotting. Phosphorylation of PLB at both Ser¹⁶ and Thr¹⁷ wassignificantly reduced by I/R, and phosphorylation at Ser¹⁶ wassignificantly recovered by ψPLB-SE. In addition, cell death-relatedCaspase 3 was activated by I/R, which was completely reversed by ψPLB-SE(FIG. 3b). Collectively, these data showed that ψPLB-SE improvedfunctional recovery after I/R, at least in part, through elevating thephosphorylation levels of PLB at Ser¹⁶.

DISCUSSION

SERCA2a is a crucial regulator of intracellular Ca²⁺ handling incardiomyocytes, and its role in heart failure and I/R injury is wellestablished [6]. Therefore, modalities that normalize SERCA2a levelsand/or activity could have significant therapeutic potentials. One suchapproach involves a gene delivery-mediated recovery of suppressedSERCA2a levels in failing hearts. This approach is effective in animalmodels of heart failure [13-16], and recently has been proven to be safeand effective in end-stage human heart failure patients [17, 18].

PLB is an endogenous inhibitor of SERCA2a and is therefore a potentialtarget for modulation of SERCA2a activity. Down-regulation of PLB withantisense RNA [35] or small interfering RNA [36] partially restoredSERCA2a activity and cardiomyocyte contractility. A dominant negativeform of PLB, K3E/R14E, designed to disrupt the structural integrity ofendogenous PLB, enhanced SERCA2a activity in neonatal and adultcardiomyocytes [35].

In this study, we showed that ψPLB-SE prevented dephosphorylation of PLBby serving as a decoy peptide for PP1. Interestingly, we found thatψPLB-TE was not as effective as ψPLB-SE (FIG. 1c). Although a structuralinsight is currently unavailable, it is possible that PLB with aphosphorylation at Ser¹⁶ is a better substrate for PP1 than PLB with aphosphorylation at Thr¹⁷. It is notable that phosphorylation at Thr¹⁷occurs only subsequent to phosphorylation at Ser¹⁶ during β-adrenergicstimulation [36]. Therefore, PLB with phosphorylation only at Thr¹⁷ maynot exist in vivo. In conclusion, we showed that IψPLB-SE elicitedcardio-protective effects by restoring SERCA2a activity. As a shortpeptide, ψPLB-SE has several therapeutic advantages over the siRNAs ormutant PLB proteins. Further experiments are warranted to determine ifψPLB-SE is effective in various surgical and genetic models of heartfailure.

Having described a preferred embodiment of the present invention, it isto be understood that variants and modifications thereof falling withinthe spirit of the invention may become apparent to those skilled in thisart, and the scope of this invention is to be determined by appendedclaims and their equivalents.

REFERENCE TO A SEQUENCE LISTING

Applicant hereby submits, in compliance with sequence rules 37 C.F.R. §§1.821-1.825, the required Sequence Listing. A copy of the SequenceListing is being submitted in computer readable format as required by 37C.F.R. §§ 1.181(e), 1.821(g), 1.825(b), or 1.825(d).

This application contains references to amino acid sequences and/ornucleic acid sequences which are being submitted concurrently herewithas the sequence listing text file 61673751_1.TXT file size 2.72KiloBytes (KB), created on 2 Feb. 2016. The aforementioned sequencelisting is hereby incorporated by reference in its entirety.

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What is claimed is:
 1. A pharmaceutical composition comprising apharmaceutically acceptable carrier and a decoy peptide or polypeptideconsisting of a peptide sequence represented by the following Formula I:X₁-Ala-X₂—X₃-Ile-Glu-X₄  (I) wherein X₁ represents 0-1 amino acidresidue(s), X₂ represents Ser, Glu, or Asp, X₃ represents Thr, Glu, orAsp and X₄ represents 0-3 amino acid residue(s), with the proviso thatX₂ is not Ser when X₃ is Thr; wherein the decoy peptide or polypeptideinhibits protein phosphatase 1 (PP1)-mediated dephosphorylation ofphospholamban (PLB) by a competitive inhibition, wherein neither X₁ norX₄ comprise membrane-spanning domains and X₁, if present, is not Tyr,and wherein the decoy peptide or polypeptide is linked to a cellmembrane-permeable peptide.
 2. The pharmaceutical composition accordingto claim 1, wherein X₁ is Arg.
 3. The pharmaceutical compositionaccording to claim 1, wherein X₄ is Met, Met-Pro, or Met-Pro-Gln.
 4. Thepharmaceutical composition according to claim 1, wherein the X₂ is Gluor Asp and X₃ is Thr, Glu, or Asp.
 5. The pharmaceutical compositionaccording to claim 1, wherein the decoy peptide or polypeptide consistsof the amino acid sequence selected from the group consisting of theamino acid sequences of SEQ ID NOs:1-6.